The change of Th subsets in lymphocytes after treatment with supernatants from RSV-infected HBECs.

<p>a. The change of Th2 cells in lymphocytes after treatment with supernatants from RSV-infected HBECs. Th2 cells mainly secreted IL-4. Monensin inhibited the secretion of newly produced cytokines in Golgi body. The positive cells in M2 which were conjugated with anti IL-4 were Th2 cells. In Fig 2.6a, the percentage of Th2 cells in lymphocytes co-cultured with RSV-infected HBECs was highest among three groups. The percentage of Th2 cells in lymphocytes co-cultured with normal HBECs was still higher than control. b: The change of Th17 cells in lymphocytes after treatment with supernatants from RSV-infected HBECs. Th17 cells mainly secreted IL-17. The positive cells in M2 which were conjugated with anti IL-17 were Th17 cells. In Fig 2.6b, the percentage of Th17 cells in lymphocytes co-cultured with RSV-infected HBECs was highest among three groups. The percentage of Th2 cells in lymphocytes co-cultured with normal HBECs was still higher than control. c: The change of Treg cells in lymphocytes after treatment with supernatants from RSV-infected HBECs. CD25 was surface marker and Foxp3 was intracellular activating factor of regulatory T cells (Treg). The positive cells in right upper region which were conjugated with anti-CD25 and anti-Foxp3 antibodies were Treg cells. In Fig 2.6c, the percentage of Treg cells in lymphocytes co-cultured with RSV-infected HBECs was lower than the other two groups. There was no difference between control group and normal HBECs group.</p

Effect of RSV-infected HBECs on the cells cycle of lymphocytes.

<p>(%, n = 8/group, mean±SEM; *P<0.01 vs L ^▵P<0.05 vs RL ^▴P<0.05 vs HL).</p

The cytopathic effects in HBECs after infection with RSV were observed under phase contrast microscopy.

<p>A. normal HBECs; B. The classical pathologic changes of RSV-infected cells: enlarged fusion cell (×100). In this vision, variant shape, shrinkage, and enhanced refraction were present in cells, as well as enlarged fusion cells and intracytoplasmic eosinophilic inclusion, which were considered to be proof of RSV infection. A fusion cell is indicated by arrows.</p

Subcellular structure of infected cells and subcellular localization of virus under electron microscopy.

<p>A is normal HBECs (×10000); B is RSV-prolonged infected HBECs in early infection stage, fissure around nucleus is seen (×10000); C is also in early infection stage, expansion of endoplasmic reticulum and a large number of lysosomes in cytoplasm were present (×15000). Both cells in B and C had double nucleoli, which indicated the cell was proliferating rapidly; D is in late infection stage, enlarged fusion cells were present (×10000). Intracellular virus particles indicated by arrows were observed both in nucleus and endoplasmic reticulum.</p

The distribution of Th subsets after treatment with supernatants from RSV-infected HBECs (%, n = 6/group, mean±SEM; *P<0.01 vs. control ^▵P<0.05 vs. normal HBECs).

<p>The percentages of Th2 and Th17 cells in RSV-infected group were higher than the other two groups, while the percentage of Treg was less than the other groups. The percentages of Th2 and Th17 cells in normal HBECs group were higher than control group.</p

Effect of RSV-infected HBECs on concentrations of IL-4, IFN-γ and IL-17 secreted by lymphocytes (pg/mL, n = 10/group, mean±SEM; *P<0.05 vs. L ^▵P<0.05 vs. RL ^▴P<0.05 vs. HL.

<p>The levels of IL-4, IL-17 and IFN-γ in group HRL were highest in four groups. The level of IFN-γ in group HL was higher than those of groups L and RL.</p

The model of RSV-infected HBECs was examined by immunofluorescence.

<p>A. Infected cells can be seen under phase contrast microscopy (×40); B. Infected cells conjugated with FITC can be seen under fluorescence microscopy (×40). Bright yellow green particles of various sizes were distributed within cytoplasm, which suggested the cells were positive for RSV infection and RSV infected model was constructed successfully.</p

Effect of RSV- infected HBECs on cell cycle of lymphocyte.

<p>Flow cytometry analysis showed significant more cells in S phase and less cells in G1 phase in group HRL compared with the other three groups. And even cells in S phase were more in group HL than the rest two groups.</p